Andrea L. Conroy

Angiopoietin-1 and angiopoietin-2 as clinically informative prognostic biomarkers of morbidity and mortality in severe sepsis

Objective:To determine the utility of angiopoietin-1 and angiopoietin-2 as potentially novel biomarkers of morbidity and mortality in patients with severe sepsis. Design:Multicenter longitudinal cohort study. Setting:Three tertiary hospital intensive care units in Hamilton, Ontario, Canada. Patients:A total of 70 patients with severe sepsis were enrolled within 24 hrs of meeting the inclusion criteria for severe sepsis and followed until day 28, hospital discharge, or death. Interventions:Clinical data and plasma samples were obtained at intensive care unit admission for all 70 patients and then daily for 1 wk and weekly thereafter for a subset of 43 patients. Levels of angiopoietin-1 and angiopoietin-2 in stored plasma samples were measured and compared with clinical characteristics, including the primary outcomes of 28-day mortality and morbidity measured by the Multiple Organ Dysfunction score. Measurements and Main Results:Lower angiopoietin-1 plasma levels (≤5.5 ng/mL) at admission were associated with increased likelihood of death (relative risk 0.49 [95% confidence interval of 0.25–0.98], p = .046). Lower angiopoietin-1 levels remained a significant predictor of 28-day mortality in a multiple logistic regression model (adjusted odds ratio of 0.282 [95% confidence interval of 0.086–0.93], p = .037). Analysis of serial data using linear mixed models confirmed that sepsis survivors had higher levels of angiopoietin-1 (p = .012) and lower daily levels of angiopoietin-2 (p = .022) than nonsurvivors. Furthermore, survivors had higher peak angiopoietin-1 levels (median 13 vs. 10 ng/mL, p = .019) and lower nadir angiopoietin-2 levels (median 2.8 vs. 6.2 ng/mL, p = .013) than nonsurvivors. A score incorporating angiopoietin-1 and angiopoietin-2 and three other markers of endothelial activation discriminated with high accuracy between fatal and nonfatal cases (c-index of 0.80 [95% confidence interval of 0.69–0.90], p < .001). Plasma levels of angiopoietin-2 correlated with clinical markers of organ dysfunction and molecular markers of endothelial cell activation. Conclusions:Angiopoietin-1 levels at admission and both angiopoietin-1 and angiopoietin-2 levels measured serially correlated with 28-day mortality in severe sepsis. Angiopoietin-2 levels also correlated with organ dysfunction/injury and a validated clinical sepsis score. These results suggest the use of angiopoietins as clinically informative biomarkers of disease severity and patient outcome in severe sepsis.

C5 deficiency and C5a or C5aR blockade protects against cerebral malaria

Experimental infection of mice with Plasmodium berghei ANKA (PbA) provides a powerful model to define genetic determinants that regulate the development of cerebral malaria (CM). Based on the hypothesis that excessive activation of the complement system may confer susceptibility to CM, we investigated the role of C5/C5a in the development of CM. We show a spectrum of susceptibility to PbA in a panel of inbred mice; all CM-susceptible mice examined were found to be C5 sufficient, whereas all C5-deficient strains were resistant to CM. Transfer of the C5-defective allele from an A/J (CM resistant) onto a C57BL/6 (CM-susceptible) genetic background in a congenic strain conferred increased resistance to CM; conversely, transfer of the C5-sufficient allele from the C57BL/6 onto the A/J background recapitulated the CM-susceptible phenotype. The role of C5 was further explored in B10.D2 mice, which are identical for all loci other than C5. C5-deficient B10.D2 mice were protected from CM, whereas C5-sufficient B10.D2 mice were susceptible. Antibody blockade of C5a or C5a receptor (C5aR) rescued susceptible mice from CM. In vitro studies showed that C5a-potentiated cytokine secretion induced by the malaria product P. falciparum glycosylphosphatidylinositol and C5aR blockade abrogated these amplified responses. These data provide evidence implicating C5/C5a in the pathogenesis of CM.

Combinations of host biomarkers predict mortality among Ugandan children with severe malaria: A retrospective case-control study

Background Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1–99.9) sensitivity and 88.8% (79.7–94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria.

Endothelium-Based Biomarkers Are Associated with Cerebral Malaria in Malawian Children: A Retrospective Case-Control Study

Background Differentiating cerebral malaria (CM) from other causes of serious illness in African children is problematic, owing to the non-specific nature of the clinical presentation and the high prevalence of incidental parasitaemia. CM is associated with endothelial activation. In this study we tested the hypothesis that endothelium-derived biomarkers are associated with the pathophysiology of severe malaria and may help identify children with CM. Methods and Findings Plasma samples were tested from children recruited with uncomplicated malaria (UM; n = 32), cerebral malaria with retinopathy (CM-R; n = 38), clinically defined CM without retinopathy (CM-N; n = 29), or non-malaria febrile illness with decreased consciousness (CNS; n = 24). Admission levels of angiopoietin-2 (Ang-2), Ang-1, soluble Tie-2 (sTie-2), von Willebrand factor (VWF), its propeptide (VWFpp), vascular endothelial growth factor (VEGF), soluble ICAM-1 (sICAM-1) and interferon-inducible protein 10 (IP-10) were measured by ELISA. Children with CM-R had significantly higher median levels of Ang-2, Ang-2:Ang-1, sTie-2, VWFpp and sICAM-1 compared to children with CM-N. Children with CM-R had significantly lower median levels of Ang-1 and higher median concentrations of Ang-2:Ang-1, sTie-2, VWF, VWFpp, VEGF and sICAM-1 compared to UM, and significantly lower median levels of Ang-1 and higher median levels of Ang-2, Ang-2:Ang-1, VWF and VWFpp compared to children with fever and altered consciousness due to other causes. Ang-1 was the best discriminator between UM and CM-R and between CNS and CM-R (areas under the ROC curve of 0.96 and 0.93, respectively). A comparison of biomarker levels in CM-R between admission and recovery showed uniform increases in Ang-1 levels, suggesting this biomarker may have utility in monitoring clinical response. Conclusions These results suggest that endothelial proteins are informative biomarkers of malarial disease severity. These results require validation in prospective studies to confirm that this group of biomarkers improves the diagnostic accuracy of CM from similar conditions causing fever and altered consciousness.

Whole blood angiopoietin-1 and -2 levels discriminate cerebral and severe (non-cerebral) malaria from uncomplicated malaria

BackgroundSevere and cerebral malaria are associated with endothelial activation. Angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) are major regulators of endothelial activation and integrity. The aim of this study was to investigate the clinical utility of whole blood angiopoietin (ANG) levels as biomarkers of disease severity in Plasmodium falciparum malaria.MethodsThe utility of whole blood ANG levels was examined in Thai patients to distinguish cerebral (CM; n = 87) and severe (non-cerebral) malaria (SM; n = 36) from uncomplicated malaria (UM; n = 70). Comparative statistics are reported using a non-parametric univariate analysis (Kruskal-Wallis test or Chi-squared test, as appropriate). Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups (UM, SM, CM), adjusting for differences due to ethnicity, age, parasitaemia and sex. Receiver operating characteristic curve analysis was used to assess the diagnostic accuracy of the ANGs in their ability to distinguish between UM, SM and CM. Cumulative organ injury scores were obtained for patients with severe disease based on the presence of acute renal failure, jaundice, severe anaemia, circulatory collapse or coma.ResultsANG-1 and ANG-2 were readily detectable in whole blood. Compared to UM there were significant decreases in ANG-1 (p < 0.001) and significant increases in ANG-2 (p < 0.001) levels and the ratio of ANG-2: ANG-1 (p < 0.001) observed in patients with SM and CM. This effect was independent of covariates (ethnicity, age, parasitaemia, sex). Further, there was a significant decrease in ANG-1 levels in patients with SM (non-cerebral) versus CM (p < 0.001). In participants with severe disease, ANG-2, but not ANG-1, levels correlated with cumulative organ injury scores; however, ANG-1 correlated with the presence of renal dysfunction and coma. Receiver operating characteristic curve analysis demonstrated that the level of ANG-1, the level of ANG-2 or the ratio of ANG-2: ANG-1 discriminated between individuals with UM and SM (area under the curve, p-value: ANG-2, 0.763, p < 0.001; ANG-1, 0.884, p < 0.001; Ratio, 0.857, p < 0.001) or UM and CM (area under the curve, p-value: ANG-2, 0.772, p < 0.001; ANG-1, 0.778, p < 0.001; Ratio, 0.820, p < 0.001).ConclusionsThese results suggest that whole blood ANG-1/2 levels are promising clinically informative biomarkers of disease severity in malarial syndromes.

Angiopoietin-2 levels are associated with retinopathy and predict mortality in Malawian children with cerebral malaria: a retrospective case-control study

Objective:To investigate the relationship among the angiopoietin–Tie-2 system, retinopathy, and mortality in children with cerebral malaria. Design:A case–control study of retinopathy-positive vs. retinopathy-negative children with clinically defined cerebral malaria. Setting:Queen Elizabeth Central Hospital in Blantyre, Malawi. Subjects:One hundred fifty-five children presenting with severe malaria and meeting a strict definition of clinical cerebral malaria (Blantyre Coma Score ⩽2, Plasmodium falciparum parasitemia, no other identifiable cause for coma) were included in the study. Interventions:None. Measurements and Main Results:Clinical and laboratory parameters were recorded at admission and funduscopic examinations were performed. Admission levels of angiopoietin-1, angiopoietin-2, and a soluble version of their cognate receptor were measured by enzyme-linked immunosorbent assay. We show that angiopoietin-1 levels are decreased and angiopoietin-2 and soluble Tie-2 levels are increased in children with cerebral malaria who had retinopathy compared with those who did not. Angiopoietin-2 and soluble Tie-2 were independent predictors of retinopathy (adjusted odds ratio [95% CI], angiopoietin-2, 4.3 [1.3–14.6], p = .019; soluble Tie-2, 9.7 [2.1–45.8], p = .004). Angiopoietin-2 and soluble Tie-2 were positively correlated with the number of hemorrhages, the severity or retinal whitening, and the extent of capillary whitening observed on funduscopic examination (p < .05 after adjustment for multiple comparisons). Angiopoietin-2 and soluble Tie-2 levels were elevated in children with cerebral malaria who subsequently died and angiopoetin-2 was an independent predictor of death (adjusted odds ratio: 3.9 [1.2–12.7], p = .024). When combined with clinical parameters, angiopoetin-2 improved prediction of mortality using logistic regression models and classification trees. Conclusions:These results provide insights into mechanisms of endothelial activation in cerebral malaria and indicate that the angiopoietin–Tie-2 axis is associated with retinopathy and mortality in pediatric cerebral malaria.

Complement Activation and the Resulting Placental Vascular Insufficiency Drives Fetal Growth Restriction Associated with Placental Malaria

Placental malaria (PM) is a major cause of fetal growth restriction, yet the underlying mechanism is unclear. Complement C5a and C5a receptor levels are increased with PM. C5a is implicated in fetal growth restriction in non-infection-based animal models. In a case-control study of 492 pregnant Malawian women, we find that elevated C5a levels are associated with an increased risk of delivering a small-for-gestational-age infant. C5a was significantly increased in PM and was negatively correlated with the angiogenic factor angiopoietin-1 and positively correlated with angiopoietin-2, soluble endoglin, and vascular endothelial growth factor. Genetic or pharmacological blockade of C5a or its receptor in a mouse model of PM resulted in greater fetoplacental vessel development, reduced placental vascular resistance, and improved fetal growth and survival. These data suggest that C5a drives fetal growth restriction in PM through dysregulation of angiogenic factors essential for placental vascular remodeling resulting in placental vascular insufficiency.

C5a Enhances Dysregulated Inflammatory and Angiogenic Responses to Malaria In Vitro: Potential Implications for Placental Malaria

Background Placental malaria (PM) is a leading cause of maternal and infant mortality. Although the accumulation of parasitized erythrocytes (PEs) and monocytes within the placenta is thought to contribute to the pathophysiology of PM, the molecular mechanisms underlying PM remain unclear. Based on the hypothesis that excessive complement activation may contribute to PM, in particular generation of the potent inflammatory peptide C5a, we investigated the role of C5a in the pathogenesis of PM in vitro and in vivo. Methodology and Principal Findings Using primary human monocytes, the interaction between C5a and malaria in vitro was assessed. CSA- and CD36-binding PEs induced activation of C5 in the presence of human serum. Plasmodium falciparum GPI (pfGPI) enhanced C5a receptor expression (CD88) on monocytes, and the co-incubation of monocytes with C5a and pfGPI resulted in the synergistic induction of cytokines (IL-6, TNF, IL-1β, and IL-10), chemokines (IL-8, MCP-1, MIP1α, MIP1β) and the anti-angiogenic factor sFlt-1 in a time and dose-dependent manner. This dysregulated response was abrogated by C5a receptor blockade. To assess the potential role of C5a in PM, C5a plasma levels were measured in malaria-exposed primigravid women in western Kenya. Compared to pregnant women without malaria, C5a levels were significantly elevated in women with PM. Conclusions and Significance These results suggest that C5a may contribute to the pathogenesis of PM by inducing dysregulated inflammatory and angiogenic responses that impair placental function.

Performance Characteristics of Combinations of Host Biomarkers to Identify Women with Occult Placental Malaria: A Case-Control Study from Malawi

Background Because of its propensity to sequester in the placental intervillous space, Plasmodium falciparum can evade detection by peripheral smear in women with placental malaria (PM). We evaluated host biomarkers as potential indicators of occult PM infections. Methods and Findings Using a case-control design, we evaluated the ability of biomarkers to identify PM in the absence of circulating peripheral parasites (n = 24) compared to placental smear-negative controls (n = 326). We measured levels of biomarkers (C3a, C5a, CRP, angiopoietin-1, angiopoietin-2, sTie-2, sEndoglin, VEGF, sFlt-1, tissue factor, and leptin) in maternal peripheral plasma at delivery. Using ROC curve analysis, we assessed the ability of clinical parameters and biomarkers to accurately detect PM infections identified by placental smear. We show that decreases in sFlt-1 and leptin and increases in CRP were associated with occult PM infections (p<0.01) and correlated with placental parasitaemia (p<0.01). Individually, all markers had moderate ability to diagnose occult PM infections with areas under the ROC between 0.62 and 0.72. In order to improve diagnostic performance, we generated simple scoring systems to identify PM infections using either a clinical score (0–2), a biomarker score (0–3) or a clinical plus biomarker score (0–5). The combinatorial model that incorporated both clinical parameters and biomarkers had an area under curve (AUC) of 0.85 (95% CI, 0.81-0.89), which was significantly better at identifying occult PM infections than the clinical score alone (p = 0.001). Conclusion These data suggest that host biomarkers in the maternal peripheral blood may improve the detection of PM in the absence of peripheral parasitaemia.

Inhaled nitric oxide for the adjunctive therapy of severe malaria: Protocol for a randomized controlled trial

BackgroundSevere malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.Methods/DesignThis prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.DiscussionNoteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.Trial RegistrationClinicalTrials.gov Identifier: NCT01255215